Objective: To investigate the effect of arsenic and its main metabolites on the apoptosis of human lung adenocarcinoma cell line A549 and the expression of pro-apoptotic genes Bad and Bik. Methods: In October 2020, A549 cells were recovered and cultured, and the cell viability was detected by the cell counting reagent CCK-8 to determine the concentration and time of sodium arsenite exposure to A549. The study was divided into NaAsO(2) exposure groups and metobol: le expoure groups: the metabolite comparison groups were subdivided into the control group, the monomethylarsinic acid exposure group (60 μmol/L) , and the dimethylarsinic acid exposure group (60 μmol/L) ; sodium arsenite dose groups were subdivided into 4 groups: control group (0) , 20, 40, 60 μmol/L sodium arsenite NaAsO(2). Hoechst 33342/propidium iodide double staining (Ho/PI) was used to observe cell apoptosis and real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression levels of Bad and Bik mRNA in cells after exposure. Western blotting was used to detect the protein expressions of Bad, P-Bad-S112, Bik, cleaved Bik and downstream proteins poly ADP-ribose polymerase PARP1 and cytochrome C (Cyt-C) , using spectrophotometry to detect the activity changes of caspase 3, 6, 8, 9. Results: Compared with the control group, the proportion of apoptotic cells in the 20, 40, and 60 μmol/L NaAsO(2) dose groups increased significantly (P<0.01) , and the expression levels of Bad, Bik mRNA, the protein expression levels of Bad, P-Bad-S112, Bik, cleaved Bik, PARP1, Cyt-C were increased (all P<0.05) , and the activities of Caspase 3, 6, 8, and 9 were significantly increased with significantly differences (P<0.05) . Compared with the control group, the expression level of Bad mRNA in the DMA exposure group (1.439±0.173) was increased with a significant difference (P=0.024) , but there was no significant difference in the expression level of Bik mRNA (P=0.788) . There was no significant differences in the expression levels of Bad and Bik mRNA in the poison groups (P=0.085, 0.063) . Compared with the control group, the gray values of proteins Bad, Bik, PARP1 and Cyt-C exposed to MMA were 0.696±0.023, 0.707±0.014, 0.907±0.031, 1.032±0.016, and there was no significant difference between the two groups (P=0.469, 0.669, 0.859, 0.771) ; the gray values of proteins Bad, Bik, PARP1 and Cyt-C exposed to DMA were 0.698±0.030, 0.705±0.022, 0.908±0.015, 1.029±0.010, and there was no difference between the two groups (P=0.479, 0.636, 0.803, 0.984) . Conclusion: Sodium arsenite induces the overexpression of Bad and Bik proteins, initiates the negative feedback regulation of phosphorylated Bad and the degradation of Bik, activates the downstream proteins PARP1, Cyt-C and Caspase pathways, and mediates the apoptosis of A549 cells.
A549 Cells ; Adenosine Diphosphate Ribose/pharmacology* ; Apoptosis ; Apoptosis Regulatory Proteins ; Arsenic ; Arsenites ; Cacodylic Acid/pharmacology* ; Caspase 3 ; Caspases/pharmacology* ; Cytochromes c/pharmacology* ; Humans ; Mitochondrial Proteins/pharmacology* ; Poisons ; Propidium/pharmacology* ; RNA, Messenger ; Sincalide/pharmacology* ; Sodium Compounds ; bcl-Associated Death Protein/metabolism*

OBJECTIVE: To explore the effects of isobavachalcone (IBC) on cell death of human breast cancer MCF-7 cells and explore the possible mechanism. METHODS: MCF-7 cells were treated with different concentrations of IBC, and the changes in cell proliferation were assessed using MTT assay. Apoptosis of MCF-7 cells following treatment with 10, 20, and 40 μmol/L IBC was analyzed using flow cytometry with annexin V-FITC/PI double staining and fluorescence microscopy, and the expressions of apoptosis- and autophagy-related proteins (Bax, Bcl-2, Akt, p-Akt, p62, and LC3) were detected with Western blotting. Electron microscopy was used to observe the changes in submicrostructure of the cells following treatment with 40 μmol/L IBC. JC-1 assay kit, ATP assay kit, and reactive oxygen species (ROS) kit were used to determine the effect of IBC on mitochondrial function of the cells. RESULTS: MTT assay showed that IBC significantly inhibited the proliferation of MCF-7 cells in a concentration- and time-dependent manner, with IC₅₀ values of 38.46, 31.31, and 28.26 μmol/L at 24, 48, and 72 h, respectively. IBC also concentration-dependently induced apoptosis of MCF-7 cells. IBC-induced cell death was inhibited by z-VAD-fmk, a caspase inhibitor (P < 0.05), but not by the necroptosis inhibitor necrostatin-1 (Nec-1). Western blotting showed that IBC-induced MCF-7 cell apoptosis by increasing Bax expression and down-regulating the expressions of Bcl-2, Akt and p-Akt-473 (all P < 0.05). With the increase of IBC concentration, the expression of autophagy-related protein p62 and the LC3-II/I ratio increased progressively. Electron microscopy revealed the presence of autophagic bodies in IBC-treated MCF-7 cells. IBC treatment also resulted in decreased mitochondrial membrane potential and intracellular ATP level and increased ROS accumulation in MCF-7 cells (P < 0.05). CONCLUSION IBC is capable of inducing both apoptosis and autophagy in MCF-7 cells, suggesting the potential value of IBC as a lead compound in the development of anti-breast cancer agents.
Adenosine Triphosphate ; Cell Death ; Chalcones ; Humans ; MCF-7 Cells ; Neoplasms ; Proto-Oncogene Proteins c-akt ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Reactive Oxygen Species/metabolism* ; bcl-2-Associated X Protein

OBJECTIVETo investigate the effects of telocinobufagin on viability and apoptosis of colorectal cancer (CRC) cells and explore the mechanism of telocinobufagin-induced apoptosis.

METHODSMTT assay was performed to detect the viability of CRC cells exposed to telocinobufagin. Nuclear staining with Hoechst 33342 and flow cytometry were used to analyze the cell death of CRC cells. Expressions of proteins related with cell apoptosis and oxidative stress were determined with Western blotting.

RESULTSTelocinobufagin decreased the viability of CRC cells in a time- and dose-dependent manner. The presence of karyopycnosis and apoptotic bodies together with the results of flow cytometry suggested that telocinobufagin induced cell apoptosis to cause cell death. Western blotting showed that telocinobufagin exposure of the cells resulted in upregulated p53 and Bax protein expressions and promoted cleavage of caspase 9 and PARP. Telocinobufagin induced phosphorylation of Bad and PARP cleavage, and suppressed phosphorylation of IKBα and TAK1 and expression of survivin in the cells.

CONCLUSIONTelocinobufagin can decrease the viability of CRC cells by inducing cell apoptosis, which involves p53-mediated Bax activation and inhibition of the IAP pathway.

Apoptosis ; Bufanolides ; pharmacology ; Caspase 9 ; metabolism ; Cell Survival ; Colorectal Neoplasms ; pathology ; Humans ; MAP Kinase Kinase Kinases ; metabolism ; NF-KappaB Inhibitor alpha ; metabolism ; Oxidative Stress ; Poly (ADP-Ribose) Polymerase-1 ; metabolism ; Tumor Cells, Cultured ; Tumor Suppressor Protein p53 ; metabolism ; bcl-2-Associated X Protein ; metabolism ; bcl-Associated Death Protein ; metabolism

OBJECTIVETo explore the molecular mechanisms by which mitochondrial estrogen receptor β (ERβ) suppresses non-small cell lung cancer cell apoptosis induced by apoptotic stimulations.

METHODSThe mitochondrial localization of ERβ in non-small cell lung cancer cell lines A549 and 201T was determined using immunofluorescence and Western blotting. The changes of apoptosis of the cells with mitochondrial ERβ overexpression or knockdown in response to cisplatin and STS treatments were assessed, and mitochondrial ERβ interaction with the pro-apoptotic protein Bad was detected using co-immunoprecipitation and Western blotting.

RESULTSERβ was localized in the mitochondria in A549 and 201T cells. ERβ overexpression significantly reduced while ERβ knockdown increased Bax activation and cell apoptosis induced by cisplatin and STS. Mitochondrial ERβ interaction with pro-apoptotic protein Bad may suppress Bax activation and its translocation to the mitochondria.

CONCLUSIONMitochondrial ERβ can suppress apoptosis of non-small cell lung cancer cells induced by cisplatin or STS through interaction with Bad, suggesting the value of mitochondrial ERβ as a new therapeutic target for treatment of non-small cell lung cancer.

Apoptosis ; Carcinoma, Non-Small-Cell Lung ; pathology ; Cell Line, Tumor ; Cisplatin ; Estrogen Receptor beta ; metabolism ; Humans ; Mitochondrial Proteins ; metabolism ; bcl-Associated Death Protein ; metabolism

Vorinostat (VOR) has been reported to enhance the cytotoxic effects of doxorubicin (DOX) with fewer side effects because of the lower DOX dosage in breast cancer cells. In this study, we investigated the novel mechanism underlying the synergistic cytotoxic effects of VOR and DOX co-treatment in cervical cancer cells HeLa, CaSki and SiHa cells. Co-treatment with VOR and DOX at marginal doses led to the induction of apoptosis through caspase-3 activation, poly (ADP-ribose) polymerase cleavage and DNA micronuclei. Notably, the synergistic growth inhibition induced by the co-treatment was attributed to the upregulation of the pro-apoptotic protein Bad, as the silencing of Bad expression using small interfering RNA (siRNA) abolished the phenomenon. As siRNA against p53 did not result in an increase in acetylated p53 and the consequent upregulation of Bad, the observed Bad upregulation was mediated by acetylated p53. Moreover, a chromatin immunoprecipitation analysis showed that the co-treatment of HeLa cells with VOR and DOX increased the recruitment of acetylated p53 to the bad promoter, with consequent bad transactivation. Conversely, C33A cervical cancer cells containing mutant p53 co-treated with VOR and DOX did not exhibit Bad upregulation, acetylated p53 induction or consequent synergistic growth inhibition. Together, the synergistic growth inhibition of cervical cancer cell lines induced by co-treatment with VOR and DOX can be attributed to the upregulation of Bad, which is induced by acetylated p53. These results show for the first time that the acetylation of p53, rather than histones, is a mechanism for the synergistic growth inhibition induced by VOR and DOX co-treatments.
Acetylation ; Antineoplastic Agents/*pharmacology ; Apoptosis/drug effects ; Cell Survival/drug effects ; Chromatin/metabolism ; Doxorubicin/*pharmacology ; Drug Synergism ; Female ; HeLa Cells ; Humans ; Hydroxamic Acids/*pharmacology ; Transcriptional Activation ; Tumor Suppressor Protein p53/genetics/*metabolism ; Uterine Cervical Neoplasms/metabolism ; bcl-Associated Death Protein/genetics/*metabolism

We have recently reported that Ginsenoside Rh2 (G-Rh2) induces the activation of two initiator caspases, caspase-8 and caspase-9 in human cancer cells. However, the molecular mechanism of its death-inducing function remains unclear. Here we show that G-Rh2 stimulated the activation of both caspase-8 and caspase-9 simultaneously in HeLa cells. Under G-Rh2 treatment, membrane death receptors Fas and TNFR1 are remarkably upregulated. However, the induced expression of Fas but not TNFR1 was contributed to the apoptosis process. Moreover, significant increases in Fas expression and caspase-8 activity temporally coincided with an increase in p53 expression in p53-non-mutated HeLa and SK-HEP-1 cells upon G-Rh2 treatment. In contrast, Fas expression and caspase-8 activity remained constant with G-Rh2 treatment in p53-mutated SW480 and PC-3 cells. In addition, siRNA-mediated knockdown of p53 diminished G-Rh2-induced Fas expression and caspase-8 activation. These results indicated that G-Rh2-triggered extrinsic apoptosis relies on p53-mediated Fas over-expression. In the intrinsic apoptotic pathway, G-Rh2 induced strong and immediate translocation of cytosolic BAK and BAX to the mitochondria, mitochondrial cytochrome c release, and subsequent caspase-9 activation both in HeLa and in SW480 cells. p53-mediated Fas expression and subsequent downstream caspase-8 activation as well as p53-independent caspase-9 activation all contribute to the activation of the downstream effector caspase-3/-7, leading to tumor cell death. Taken together, we suggest that G-Rh2 induces cancer cell apoptosis in a multi-path manner and is therefore a promising candidate for anti-tumor drug development.
Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Caspase 8 ; metabolism ; Caspase 9 ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cytochromes c ; metabolism ; Enzyme Activation ; drug effects ; Ginsenosides ; chemistry ; pharmacology ; HeLa Cells ; Humans ; Inhibitory Concentration 50 ; Mitochondria ; drug effects ; metabolism ; Protein Transport ; drug effects ; Receptors, Death Domain ; metabolism ; Receptors, Tumor Necrosis Factor, Type I ; metabolism ; Signal Transduction ; drug effects ; Tumor Suppressor Protein p53 ; metabolism ; Up-Regulation ; drug effects ; bcl-2 Homologous Antagonist-Killer Protein ; metabolism ; bcl-2-Associated X Protein ; metabolism ; fas Receptor ; metabolism

PURPOSE: Lactobacillus casei (L. casei) is known to exert anti-proliferation effects on many types of cancer cells. However, the effect of L. casei on liver cancer has not been reported. Accordingly, the aim of this study was to determine the anti-cancer effect of L. casei extract on Huh7 cells. MATERIALS AND METHODS: L. casei ATCC393 extract was prepared and purified. After the treatment of L. casei extract on Huh7 cells, cell viability, cell cycle arrest and cell death were analyzed by flow cytometry. The expression levels of tumor necrosis factor-alpha receptor 1 (TNFR1) and death receptor 3 (DR3) mRNA related with extrinsic apoptosis were assessed by reverse transcription polymerase chain reaction. Additionally, P21 and P27 cell cycle proteins as well as Caspase-3, -8, -9, phospho-Bad and Bcl-2 apoptosis proteins were analyzed by western blot analysis. To determine the effect of L. casei extract on cancer stem-like cells, we analyzed changes in side population fraction through flow cytometry. RESULTS: The cell viability of Huh7 cells treated with L. casei extract was decreased by 77%, potentially owing to increases in the rates of Huh7 cells arrested in the G2/M phase (3% increase) and that underwent apoptosis (6% increase). The expression levels of TNFR1 and DR3 mRNA, as well as P21 and P27 cell cycle proteins, were increased. Meanwhile, the expressions of caspase-8, -9, phospho-Bad and Bcl-2 proteins decreased. However, in the case of side population cells, no remarkable changes were observed. CONCLUSION: L. casei extract exerts a potent anti-tumor effect on the viability of liver cancer cells, although not on cancer stem-like cells.
Apoptosis/drug effects ; Carcinoma, Hepatocellular/*pathology ; Caspase 8/metabolism ; Caspase 9/metabolism ; Cell Cycle Checkpoints/drug effects ; Cell Extracts/*pharmacology ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Cyclin-Dependent Kinase Inhibitor p21/metabolism ; Cyclin-Dependent Kinase Inhibitor p27/metabolism ; Cytostatic Agents/*pharmacology ; Flow Cytometry ; Gene Expression Regulation, Neoplastic/drug effects ; Humans ; Lactobacillus casei/*chemistry ; Liver Neoplasms/*pathology ; Proto-Oncogene Proteins c-bcl-2/metabolism ; RNA, Messenger/metabolism ; Receptors, Tumor Necrosis Factor, Member 25/metabolism ; Receptors, Tumor Necrosis Factor, Type I/metabolism ; bcl-Associated Death Protein/metabolism

OBJECTIVESTo prove the protective effect of Edaravone to neurons and to study the particular mechanism.

METHODSNeurons were collected from 18-day fetal rat brains and a culture of almost pure neurons was obtained after 14-day culture, then the cells were randomly assigned to one of the three groups: control group, hydrogen peroxide (H₂O₂)-treated group, and Edaravone-treated group. In H₂O₂-treated group, 300 µmol/L H₂O₂ was added to the medium, followed by returning to the normal culture for the presupposition of time. In Edaravone-treated group, 500 µmol/L Edaravone was prophylactically added to the medium for 30 minutes before the insult. Morphology of mitochondria was visualized by transmission electron microscopy. The rate of apoptotic cells was detected by flow cytometry analysis. The relationships between the proteins and the key proteins expressions were observed by immunoprecipitation and immunoblotting.

RESULTSCompared to the Edaravone-treated group, mitochondria in H₂O₂-treated group displayed more vesicular matrix compartments at the same time. Percentage of apoptotic cells in H₂O₂-treated group after 0.5, 2, 6 and 12 h were 14.40% ± 1.23%, 45.50% ± 2.81%, 56.40% ± 3.53%, 62.50% ± 4.23%, which were higher than control group (F = 274.8, P < 0.01). Edaravone-treated group were 0.90% ± 0.07%, 1.10% ± 0.08%, 3.50% ± 1.90%, 12.60% ± 1.10%, which were lower than H₂O₂-treated group (F = 362.7, P < 0.01). After H₂O₂ stimulation for 0.5 h in H₂O₂-treated group, the levels of p-JNK (Thr183/Tyr185) and cytochrome c in cytosol and BAX in heavy membrane were increased significantly at 0.5 h, reaching a peak at 12 h after stimulation, In addition, the expressions of p-BAD, BAX, BAD and 14-3-3 of cytoplasm decreased, however, these changes were inhibited in the Edaravone-treated group.

CONCLUSIONSAs a free radical scavenger, the Edaravone could protect neurons by inhibiting the activity of JNK, the disassociation of BAD from 14-3-3 and the translocation of BAX from the cytosol to mitochondria.

14-3-3 Proteins ; metabolism ; Animals ; Antipyrine ; analogs & derivatives ; pharmacology ; Apoptosis ; drug effects ; Cells, Cultured ; Free Radical Scavengers ; pharmacology ; Hydrogen Peroxide ; metabolism ; MAP Kinase Signaling System ; Mitochondria ; drug effects ; Neurons ; drug effects ; Neuroprotective Agents ; pharmacology ; Primary Cell Culture ; Rats ; Rats, Sprague-Dawley ; bcl-2-Associated X Protein ; metabolism ; bcl-Associated Death Protein ; metabolism

PURPOSE: To investigate the anti-tumor effect of capsaicin on human pharyngeal squamous carcinoma cells (FaDu). MATERIALS AND METHODS: The expression of apoptosis/cell cycle-related proteins (or genes) was examined by reverse transcriptase-polymerase chain reaction, western blotting and ELISA methods, while the apoptotic cell population, cell morphology and DNA fragmentation levels were assessed using flow cytometry, fluorescence microscopy and agarose gel electrophoresis. RESULTS: Capsaicin was found to inhibit the growth and proliferation of FaDu cells in a dose- and time-dependent manner. Apoptotic cell death was confirmed by observing increases in nuclear condensation, nuclear DNA fragmentation and sub-G1 DNA content. The observed increase in cytosolic cytochrome c, activation of caspase 3 and PARP (p85) levels following capsaicin treatment indicated that the apoptotic response was mitochondrial pathway-dependent. Gene/protein expression analysis of Bcl-2, Bad and Bax further revealed decreased anti-apoptotic Bcl-2 protein and increased pro-apoptotic Bad/Bax expression. Furthermore, capsaicin suppressed the cell cycle progression at the G1/S phase in FaDu cells by decreasing the expression of the regulators of cyclin B1 and D1, as well as cyclin-dependent protein kinases cdk-1, cdk-2 and cdk-4. CONCLUSION: Our current data show that capsaicin induces apoptosis in FaDu cells and this response is associated with mitochondrial pathways, possibly by mediating cell cycle arrest at G1/S.
Apoptosis/drug effects ; Blotting, Western ; Capsaicin/*pharmacology ; Carcinoma, Squamous Cell/*metabolism ; Cell Cycle/drug effects ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Humans ; Microscopy, Fluorescence ; Pharyngeal Neoplasms/*metabolism ; Proto-Oncogene Proteins c-bcl-2/genetics/metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; bcl-2-Associated X Protein/genetics/metabolism ; bcl-Associated Death Protein/genetics/metabolism

Adult ; Altitude Sickness ; metabolism ; pathology ; Apoptosis ; BH3 Interacting Domain Death Agonist Protein ; metabolism ; Erythroblasts ; cytology ; pathology ; Humans ; Male ; Middle Aged ; RNA, Messenger ; genetics ; bcl-2-Associated X Protein ; metabolism ; bcl-X Protein ; metabolism